Recent studies suggest that H2O2, at subtoxic concentrations generated in response to the activation of a variety of cell surface receptors, functions as an intracellular messenger. However, the intracellular targets of H2O2 action have not been identified. A procedure to detect proteins with reactive cysteine residues susceptible to oxidation by intracellularly generated H2O2 is now described. This approach is based on the labeling of proteinaceous cysteine with 5-iodoacetamidofluorescein at pH 5.5 and immunoblot analysis of the labeled proteins with antibodies specific to fluorescein. With this procedure, many proteins in human A431 cells were shown to contain reactive cysteines and to be readily oxidized by H2O2 generated in response to cellular stimulation with epidermal growth factor. One of these H2O2-sensitive proteins was identified as protein tyrosine phosphatase 1B.